Vigna Unguiculata (L.) Walp.]

Plant Reprod (2016) 29:165–177 DOI 10.1007/s00497-015-0273-3 ORIGINAL ARTICLE New observations on gametogenic development and reproductive experimental tools to support seed yield improvement in cowpea [Vigna unguiculata (L.) Walp.] 1 2 1 Rigel Salinas-Gamboa • Susan D. Johnson • Nidia Sańchez-Leoń • 2 1 Anna M. G. Koltunow • Jean-Philippe Vielle-Calzada Received: 28 October 2015 / Accepted: 21 December 2015 / Published online: 4 January 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com Key message Cowpea reproductive tools. floral bud size and reproductive organ development, Abstract Vigna unguiculata L. Walp. (cowpea) is recog- determining that gametogenesis occurs more rapidly in the nized as a major legume food crop in Africa, but seed anther than in the ovule. We also show that the mode of yields remain low in most varieties adapted to local con- megagametogenesis is of the Polygonum-type and not ditions. The development of hybrid cowpea seed that could Oenothera-type, as previously reported. Finally, we be saved after each generation, enabling significant yield developed a whole-mount immunolocalization protocol increases, will require manipulation of reproductive and applied it to detect meiotic proteins in the cowpea development from a sexual to an asexual mode. To develop megaspore mother cell, opening opportunities for com- new technologies that could support the biotechnological paring the dynamics of protein localization during male manipulation of reproductive development in cowpea, we and female meiosis, as well as other reproductive events in examined gametogenesis and seed formation in two this emerging legume model system. transformable, African-adapted, day-length-insensitive varieties. Here, we show that these two varieties exhibit Keywords Megagametogenesis Á Megasporogenesis Á distinct morphological and phenological traits but share a Microgametogenesis Á Cowpea Á Reproductive calendar Á common developmental sequence in terms of ovule for- Whole-mount immunolocalization Á Ovule mation and gametogenesis. We present a reproductive calendar that allows prediction of male and female gametogenesis on the basis of sporophytic parameters related to Introduction Many of the poorest countries in the world derive 10–20 % Communicated by Dolf Weijers. of their total dietary protein from grain legumes, members of the Fabaceae (Akibode and Maredia, 2011). Cowpea, Anna M. G. Koltunow and Jean-Philippe Vielle-Calzada are co-senior Vigna unguiculata (L.) Walp. is one of the eight grain authors. legumes currently being targeted for yield and agronomic Electronic supplementary material The online version of this improvement by the Consultative Group for International article (doi:10.1007/s00497-015-0273-3) contains supplementary Agricultural Research (CGIAR). Cowpea originated in material, which is available to authorized users. Africa, which produces over 95 % of the 5.4 million tons & Jean-Philippe Vielle-Calzada of dried cowpeas produced globally, on an estimated 11.5 [email protected] million hectares of land across a diverse range of farming 1 systems, in the semiarid regions of West and Central Grupo de Desarrollo Reproductivo y Apomixis, Laboratorio Africa. Approximately 200 million individuals are esti- Nacional de Geno´mica para la Biodiversidad, CINVESTAV Irapuato, CP 36821 Guanajuato, Mexico mated to consume the grain daily in West Africa as it provides a high source of protein (25 % by weight) and all 2 Agriculture Flagship, Commonwealth Scientific and Industrial Research Organization, plant organs are edible. Cowpea is an important component Private Bag 2, Glen Osmond, SA 5064, Australia in increasing sustainable farming production by being 123 166 Plant Reprod (2016) 29:165–177 drought resistant and having the ability to fix atmospheric megagametogenesis in Vigna. An important prerequisite nitrogen, contributing to reduced soil erosion. for developing robust hybrid seed production systems or Conventional cowpea breeding is currently underpinned attempting to alter reproduction in cowpea is a clear by the use of molecular markers (Lucas et al. 2011, 2013), understanding of the temporal and spatial events of sexual correlations between genetic and physical maps (Pottorff male and female gametophyte formation in relation to et al. 2014), and large-scale genomic sequencing (Andargie flower morphology and supporting cytological tools to et al. 2011; Barrera-Figueroa et al. 2011; Huang et al. efficiently and accurately examine changes induced by 2012). Transformation technologies have been established hybridization, mutation, or transgenic approaches. for cowpea and are being implemented to accelerate In this paper, we applied a range of cytological techniques agronomic performance, with strong indications of success to a pair of cultivated African cowpea varieties of known in the development of seed borer-resistant lines (Kang et al. pedigree developed by the International Institute of Tropical 2014; Popelka et al. 2006). Despite advances in increasing Agriculture (Kang et al. 2014) and developed a reproductive cowpea yields and stress tolerance over the last two dec- calendar that allows prediction of male and female game- ades, many varieties are still low yielding and susceptible togenesis on the basis of sporophytic parameters such as to a range of biotic and abiotic stresses (Lush and Evans floral bud size, gynoecium length, and integument growth. 1981; Singh 2014; Huynh et al. 2015; Lucas et al. 2015). Although one flowers earlier than the other, both share a The development of cowpea hybrids has the potential to common developmental sequence in terms of ovule forma- increase crop yields by 30 % as suggested by published tion and gametogenesis. Contrary to previous reports, we studies aimed at identifying levels of heterosis in progeny show that the female gametophyte is not of the Oenothera - from crosses between inbred cowpea parents (Bhaskaraiah type but consistently develops from three mitotic nuclear et al. 1980; Ortiz 1998; Ushakumari et al. 2010; Kumari divisions of the most chalazal megaspore, giving rise to a et al. 2012). Hybrid seed production systems have not yet megagametophyte of the Polygonum-type. Callose staining been developed in cowpea. Impediments include self-fer- of pollen tubes and tracking of sperm cells within the tilizing hermaphrodite flowers, low outcrossing rates megagametophyte showed that fertilization occurs 12–14 h (\5 %; Timko and Singh 2008), and high flower drop rates after pollination. We also developed a simple whole-mount (Wien and Summerfield 1980; Ehlers and Hall 1996; immunolocalization procedure with sufficient sensitivity to Ojehomon and Samyaolu 1970). In all current hybrid seed allow the detection of meiotic proteins specifically expressed production systems, hybrid seeds need to be generated and in the megaspore mother cell. Collectively, the methods purchased each growing season, impeding their purchase developed here for cowpea are likely to be applicable to by poor farmers. The current inability to produce hybrid analyses of gametogenesis in a range of legumes. seeds relates to the plant’s sexual reproductive process. Meiosis and recombination during gamete formation and gamete fusion at fertilization alleviate heterosis, concur- Materials and methods rently leading to trait segregation. Development of cowpea hybrids from which seeds could be saved by farmers would Plant material and growth conditions offer the potential to generate higher-yielding cowpea crops in an economical manner. The generation of such Seeds of Vigna unguiculata IT97K-499-35 and IT86D- ‘‘self-reproducing’’ hybrids would require changing 1010 were germinated and grown in either a soil mixture of reproduction in the sexually reproducing hybrid to an 3:1:1 sunshine soil/vermiculite/perlite (v/v/v), and Osmo- asexual seed forming mode mimicking the events of apo- cote (1.84 kg/m3) as a fertilizer, or a mixture of BioGrow mixis to generate clonal seed where vigor was preserved. (60 % coarse milled composted pine bark[7 mm, 30 % of Recent studies have provided proof-of-concept of clonal fine milled composted pine bark 3–6 mm, and 10 % of seed generation, which might be applicable for the gener- washed coarse river sand) and Osmocote (1.84 kg/m3). ation of self-reproducing cowpea hybrids (Marimuthu et al. Both varieties were grown under greenhouse condi- 2011; Hand and Koltunow 2014; Gilbert 2015). tions but did not flower during winter; temperatures in While general descriptions of reproductive development Irapuato were 25–28 °C (spring–summer) and 20–24 °C in members of the Fabaceae have been published from a (autumn–winter), and temperatures in Adelaide were physiological and developmental perspective (Brown 1917; 22–28 °C (summer) and 18–23 °C (winter). Mitchel 1975; Rembert 1977; Albertsen and Palmer 1979; Kennell and Horner 1985; Moço and Mariath 2003; Cytological analysis Soverna et al. 2003; Rodriguez-Rianõs et al. 2006; Chehregani and Tanaomi 2010; Ghassempour et al. 2011), For light microscopy analysis, floral buds were manually there is surprisingly scarce information on micro- and dissected and fixed in FAA (ethanol (50 %), formaldehyde 123 Plant Reprod (2016) 29:165–177 167 (10 %), acetic acid (4 %), and water) for at least 12 h, Whole-mount protein immunolocalization before being dissected with hypodermic needle and scalpel. Both the bud and the gynoecia length were measured Immunolocalization was performed as previously descri- before proceeding

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